肠道病毒A71型结构蛋白和非结构蛋白对p85表达及p38途径活化的影响  

Effects of Enterovirus-A71 Structural and Non-structural Proteins on Expression of p85 and Activation of p38 Pathway

作　　者：孙萍萍 王佳慧 杨世昭 任晴 王蒋丽[2] 谢广成 SUN Ping-ping;WANG Jia-hui;YANG Shi-zhao;REN Qing;WANG Jiang-li;XIE Guang-cheng(School of Basic Medicine,Chengde Medical University,Chengde,Hebei,067000,China;Chengde Center£srDisease Control and Prevention,Chengde,Hebei,067000,China)

机构地区：[1]承德医学院基础医学院,河北承德067000 [2]承德市疾病预防控制中心

出　　处：《承德医学院学报》2022年第3期181-186,共6页Journal of Chengde Medical University

基　　金：国家自然科学基金(81702008);河北省自然科学基金(H2018406024);承德医学院科研项目(KY2020002);承德医学院国家级大学生创新创业训练计划项目(2021001);河北省科技厅“技术创新引导专项-科技工作会商”项目;河北省高校重点学科(冀教高[2013]4号)。

摘　　要：目的探讨肠道病毒A71型(EV-A71)结构蛋白和非结构蛋白对p85表达和p38途径活化的影响。方法通过RT-PCR扩增EV-A71的12个基因片段,经酶切反应和连接反应将目的片段连接到pcDNA3.1(+)/myc-His A真核表达载体,制备去内毒素的重组质粒;使用SuperFect转染试剂将2μg重组真核质粒转染至HEK293细胞24h后,免疫印迹检测p85表达和p38的磷酸化。结果正确构建12个EV-A71结构蛋白和非结构蛋白的真核重组质粒;与未转染细胞相比,EV-A71 P1区结构蛋白(VP1、VP3和VP4)、P2区非结构蛋白(2A、2B、2C和2BC)和P3区非结构蛋白(3A、3C、3D和3AB)均能不同程度的改变p85的表达和p38的磷酸化水平;EV-A71结构蛋白VP2虽不能显著提升p85的表达,但能提升p38的磷酸化水平。结论EV-A71的12个亚单位蛋白可与宿主细胞发生互作,进而调控宿主细胞的生物学过程。Objective To explore the effects of enterovirus-A71(EV-A71)structural and non-structural proteins on expression of p85 and activation of p38 pathway.Methods Twelve gene segments of EV-A71 were amplified by RT-PCR.The target fragments were ligated into the pcDNA3.1(+)/myc-His A eukaryotic expression vector after double digestion by restriction enzymes and ligation reaction,to prepare the endotoxin-free recombinant plasmid.Twelve recombinant plasmids were transfected into HEK293 cells using SuperFect transfection reagent for 24h after endotoxin-free plasmids were prepared.The dose of each plasmid per well was 2μg.Expression of p85 and phosphorylation level of p38 were detected through western blotting.Results Twelve recombinant eukaryotic expression plasmids of EV-A71 structural and non-structural proteins were constructed successfully.Compared with untransfected cells,the expression of p85 and the phosphorylation level of p38 were regulated by EV-A71 structural proteins in P1 region(VP1,VP3 and VP4),non-structural proteins in P2 region(2A,2B,2C and 2BC)and non-structural proteins in P3 region(3A,3C,3D and 3AB)in different degrees.Although EV-A71 structural protein VP2 could not significantly elevate the expression of p85,the phosphorylation level of p38 was increased.Conclusion Twelve subunit proteins of EV-A71 interact with host cells,which further regulate the biological process of host cells.

关 键 词：肠道病毒A71型 亚单位蛋白 P85 p38途径 

分 类 号：R373.24[医药卫生—病原生物学]